Molecular modeling and co-expression analysis of human stem cell factor as fusion partner to granulocyte colony stimulating factor for improving their bioactivity.

Bijayalaxmi Sahoo, Sagarika Dash, Srimeenakshi Sankarnarayanan, Balaram Mishra, Praveen Kumar Guttula, Rakesh Bhaskar, Mukesh Kumar Gupta

Journal of biomolecular structure & dynamics 2021 Sep

filter terms:

Human granulocyte colony stimulating factor (hG-CSF) is an expensive hematopoietic growth factor that is clinically used in human for the treatment of neutropenia in diseases such as AIDS, aplastic anemia, myelodysplastic syndrome and congenital or chemotherapy-induced neutropenia. Here, through a computational biology approach, we show that human stem cell factor (hSCF) could be a better fusion partner than human thyroid peroxidase (hTPO), human erythropoietin (hEPO) and human interleukin-3 (hIL3) for co-expression with hG-CSF. Molecular modeling of hG-CSF-hSCF fusion protein with hG-CSF and hSCF receptors showed that binding of fusion protein with human granulocyte colony stimulating factor receptor (hG-CSFR) did not inhibit its binding to human stem cell factor receptor (hSCFR) and vice versa. To validate the results, coding sequences of hG-CSF and hSCF were cloned and co-expressed as fusion protein and their bioactivity was evaluated on hG-CSF responsive 3T3 cell line. The fused expression vector expressed recombinant hG-CSF-hSCF upon IPTG-induction, as revealed by real-time polymerase chain reaction (RT-PCR), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Bioactivity analysis confirmed that rhG-CSF-hSCF protein had higher bioactivity than hG-CSF. Thus, hSCF could be a good fusion partner for hG-CSF and its co-expression as hG-CSF-hSCF may offer an alternative to individual use of two hematopoietic factors in clinics. Future studies should determine the purification strategies, folding status and mechanism of action of the recombinant proteins. Communicated by Ramaswamy H. Sarma.

Citation

Bijayalaxmi Sahoo, Sagarika Dash, Srimeenakshi Sankarnarayanan, Balaram Mishra, Praveen Kumar Guttula, Rakesh Bhaskar, Mukesh Kumar Gupta. Molecular modeling and co-expression analysis of human stem cell factor as fusion partner to granulocyte colony stimulating factor for improving their bioactivity. Journal of biomolecular structure & dynamics. 2021 Sep;39(14):4990-5004